Tyrosin Kinase Inhibition Restores the Membrane Localization of FLT3-ITD

Abstract

The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regards to oncogenic alterations that do not only serve as prognostic markers but also as targets for therapy in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Due to sequence-dependent protein conformation changes, FLT3-ITD tends to auto-phosphorylate and displays a constitutive intracellular localization. As tyrosine kinase inhibitors (TKIs) efficiently block FLT3 auto-phosphorylation, we set out to study the effect of TKI on the subcellular localization of the FLT3 receptor and its mutants.We visualized FLT3 WT and its mutants (N676K, D835Y, W51 (ITD), NPOS (ITD), K644R, W51-K644R) in transiently transfected U2OS cells, using anti-FLT3 immunofluorescence in combination with confocal microscopy. Treatment with 50nM of the TKI AC220 for 6 hours restored the membrane localization of both ITD (Figure 1 A) and D835Y. The TKI-induced surface expression of FLT3-ITD was confirmed by flow cytometry (mean fluorescence intensity, MFI) of BaF3 cells stably expressing FLT3 WT or mutants and in the homozygous (ITD/ITD) AML cell line (MV4-11). Similar results were obtained in cells from an AML patient derived xenograft (pdx) with FLT3-ITD and loss of heterozygosity (LOH) (Figure 1 B). Western blot analysis showed increased glycosylation of FLT3-ITD after TKI-treatment (Figure 1 C) suggesting that auto-phosphorylation prevents physiological processing of the receptor, which is required for maturation and surface expression. To monitor the subcellular localization of FLT3 and its mutants in vivo we have generated a FLT3-GFP fusion. Proliferation assays in BaF3 cells confirmed that the C-terminal GFP-tag does not alter the function of FLT3. Upon FLT3-ligand (FL) stimulation of FLT3-GFP we observed FLT3-internalization by life cell imaging in U2OS cells. Conversely, TKI treatment increased cell surface expression of FLT3-GFP.Our findings have translational implications when considering FLT3 as a target for immunotherapy in AML, since FLT3 surface expression might facilitate antigen recognition. Especially FLT3-ITD positive patients with LOH may benefit from a combined TKI-immunotherapy approach. Therefore, we currently study the effect of AC220 on FLT3 antibody-dependent cellular cytotoxicity (ADCC), mediated by natural killer (NK) cells. Taken together, we discovered that TKI alters the subcellular localization and protein processing of specific FLT3 mutants. However, the complex interplay between the individual posttranslational modifications and their role in FLT3 traffic remains elusive. [Display omitted] DisclosuresKrupka:AMGEN Research (Munich): Research Funding. Subklewe:AMGEN Research (Munich): Research Funding.